Carboxymethyl chitosan prolongs adenovirus‐mediated expression of IL‐10 and ameliorates hepatic fibrosis in a mouse model

Abstract Effective and safe liver‐directed gene therapy has great promise in treating a broad range of liver diseases. While adenoviral (Ad) vectors have been widely used for efficacious in vivo gene delivery, their translational utilities are severely limited due to the short duration of transgene expression and solicitation of host immune response. Used as a promising polymeric vehicle for drug release and nucleic acid delivery, carboxymethyl chitosan (CMC) is biocompatible, biodegradable, anti‐microbial, inexpensive, and easy accessible. Here, by exploiting its biocompatibility, controlled release capability and anti‐inflammatory activity, we investigated whether CMC can overcome the shortcomings of Ad‐mediated gene delivery, hence improving the prospect of Ad applications in gene therapy. We demonstrated that in the presence of optimal concentrations of CMC, Ad‐mediated transgene expression lasted up to 50 days after subcutaneous injection, and at least 7 days after intrahepatic injection. Histologic evaluation and immunohistochemical analysis revealed that CMC effectively alleviated Ad‐induced host immune response. In our proof‐of‐principle experiment using the CCl4‐induced experimental mouse model of chronic liver damage, we demonstrated that repeated intrahepatic administrations of Ad‐IL10 mixed with CMC effectively mitigated the development of hepatic fibrosis. Collectively, these results indicate that CMC can improve the prospect of Ad‐mediated gene therapy by diminishing the host immune response while allowing readministration and sustained transgene expression.

Liver is a key organ in the human body and carries out a variety of essential functions including digestion, metabolism, detoxification, immunity and blood clotting. 1 At the cellular level, hepatocytes constitute the vast majority of cells in the liver parenchyma and are implicated in the majority of monogenic liver inherited disorders, metabolic disorders, viral infections, and malignancies. [1][2][3] Even though conventional treatments can alleviate symptoms of some liver disorders, very few curative treatments currently exist. 1 Therefore, effective and safe liver-directed gene therapy holds great promise in treating a broad range of liver diseases, such as cancer, metabolic disorders, and certain monogenic disorders [1][2][3] .
Liver is an ideal target for gene therapy since it is one of the largest organs in the human body, and contains 10%-15% of the total blood volume of the body. For the past several decades, numerous efforts have been devoted to the development of liver-directed gene delivery systems. [1][2][3] The gene delivery systems are in general divided into two categories: viral vector-based and non-viral vector-based delivery systems. The viral vector systems include a large group of recombinant, replication-deficient viruses; and the most commonly-used ones are adenoviral (Ad) vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, as well as certain less frequently used ones such as foamy viral vectors and herpes simplex viral vectors. [3][4][5][6][7][8] While viral vectors are highly effective in gene delivery and have been used in approximately 70% clinical trials, the major hurdles for viral vectors include potential carcinogenesis and/or immunogenicity. Non-viral vector-based delivery systems take advantage of receptor-mediated endocytosis and/or membrane fusion functions via the use of lipids, polymers, proteins, and peptides, as well as by physical forces such as needle injection, gene gun, electroporation, sonoporation, and hydrodynamic delivery. While the major challenge for non-viral vector-mediated gene delivery is its relatively low efficiency, an increasing number of non-viral vectors are emerging as valid vehicles for the delivery of genetic materials, especially the use of lipid-based nanoparticles for RNA delivery.
Nonetheless, viral vectors remain as one of the most preferred approaches to target hepatocytes. Among them, Ad vector represents the prototype viral vector with high gene transfer efficiency, wellunderstood virology and pathogenicity, and ease to mass production. 6,7 The major drawbacks for Ad vector as a liver-targeted delivery system are a relatively short-term of transgene expression and the solicitation of host immune response. Therefore, it is conceivable that overcoming the above shortcomings should make Ad vector a more desirable gene delivery vector for liver-directed gene therapy.
As a product of the deacetylation of chitin, chitosan is a linear polysaccharide, multi-functional, and eco-friendly antifouling polymer. [9][10][11][12] Chitosan is nontoxic, biocompatible, and biodegradable, which makes it a polymer of choice for many biomedical and pharmaceutical applications since it was first reported in late 1990s. 12 The chemical versatility of chitosan and its derivatives is reflected by its ability to form a poly-cationic charged polymer at physiological pH, and by its modifiable molecular weight. 12 The chemical and biological properties of chitosan are dependent on the degree of deacetylation, polymeric molecular weight, and types of surface modifications. 12 Several features make chitosan and its derivatives promising carriers for gene delivery including biocompatibility, biodegradability, nontoxicity, antimicrobial activity, low immunogenicity, low cost, and easy accessibility. 9,[11][12][13] The carboxymethylation of chitosan yields carboxymethyl chitosan (CMC) with superior solubility in physiological pH, increased antimicrobial and anti-inflammatory activity, and easier cellular uptake. 14,15 In fact, CMC has been commercialized with a wide range of applications in biomedical fields, such as wound healing, biological imaging, tissue engineering, and drug delivery. 10

| Cell culture and chemicals
HEK-293 derivative 293pTP and RAPA cells were previously characterized and used for adenovirus packaging and subsequent amplification. 17,18 These cells were cultured in high glucose complete Dulbecco's modified Eagle's medium supplemented with 10% FBS (Cat# S711-001S, Lonsera), 100 units of penicillin, and 100 μg of streptomycin at 37 C in 5% CO 2 as described. 19 2.2 | Construction, amplification, and purification of the recombinant adenoviruses Ad-FLuc, Ad-IL10, and Ad-GFP All recombinant adenoviruses were constructed by using the AdEasy technology. 24 The Ad-FLuc, which co-expresses firefly luciferase (FLuc) and GFP, was generated as described in our previous studies. [25][26][27] For the construction of Ad-IL10, the coding region of human IL-10 was PCR amplified and subcloned into an adenoviral shuttle vector, pAdTrack-CMV. The resultant vector was used to generate the recombinant adenoviral plasmid pAd-IL10 through homologous recombination with an adenoviral backbone vector in bacterial BJ5183 cells. 24,28,29 The adenoviral plasmid pAd-IL10 was subsequently linearized and used to generate recombinant adenovirus Ad-IL10 in 293pTP or RAPA packaging cells.
The Ad-IL10 also co-expresses GFP as a marker for tracking infection efficiency as described. 30, 31 An analogous adenovirus expressing GFP only, Ad-GFP, was used as a mock virus control. [32][33][34] For direct in vivo injection studies, Ad-FLuc, Ad-IL10, and Ad-GFP were amplified in large scale and purified through CsCl gradient ultracentrifugation, as described. 24,35,36 The purified high titer adenovirus stocks were titered, aliquoted, and stored at À20 C. Desalting dialysis against sterile PBS was performed immediately prior to use. Subcutaneous or intrahepatic injection of Ad-GFP mixed with or without CMC was carried out to assess Ad-induced host immune response. For the acute response, two routes of Ad delivery were assessed. In the first route, C57BL/6J mice (n = 9, male, 6-week old) were subcutaneously injected into both flanks with 10 10 pfu (plaque forming unit) of Ad-GFP mixed with either PBS alone or 1.3% CMC (wt/vol, in PBS) in 30 μl total volume. Three mice were sacrificed at 6, 24, and 72 h after injection. The injection sites were retrieved for histologic and IHC analyses. In the second route, C57BL/6J mice (n = 30, male, 6-week old) were subjected to intrahepatic injection with 10 10 pfu of Ad-GFP mixed with either PBS alone (Ad-GFP, control group) or

| Subcutaneous and intrahepatic injection of Ad-FLuc encapsulated with CMC
Subcutaneous injection of the adenovirus Ad-FLuc was used to determine the effect of various concentrations of CMC on adenovirusmediated transgene expression as described. 37 Specifically, C57BL/6J mice (male, 6-week old, n = 4) were subcutaneously injected into both flanks with 10 10 pfu of Ad-FLuc, which was premixed with 0% (wt/vol, in PBS), 1.25% (wt/vol, in PBS), 2.5% (wt/vol, in PBS), and 5% (wt/vol, in PBS) CMC (in 30 μl total volume) at each injection site, as shown in Figure 1a. Whole body optical bioluminescence imaging was performed at 3,9,15,21,28,35,43, and 50 days after injection by using the IVIS Spectrum In Vivo Imaging System (Perkin Elmer) with D-Luciferin potassium (Gold Biotechnology, Inc.) as luciferase substrate as described. 38,39 For intrahepatic injection of Ad-FLuc, C57BL/6J mice (male, 6-week old, n = 4 each group) received intrahepatic injection of 10 10 pfu of Ad-FLuc, which was premixed with 0% CMC (wt/vol, in PBS), or 1.3% CMC (wt/vol, in PBS), all in 30 μl total volume, as shown in Figure S1. Whole body optical bioluminescence imaging was performed at 3, 7, and 12 days after injection by using the IVIS Spectrum in vivo imaging system as described. [40][41][42] 2.5 | Establishment of the mouse model of chronic hepatic injury and fibrosis The hepatic fibrosis model was established as described. 43 Briefly, C57BL/6J mice (male, 6-week old) were intraperitoneally injected with 2.0 μl/g body weight (g.b.w.) of 20% CCl 4 solution (wt/vol, in olive oil) twice a week for up to 8 weeks as described. 43 The control mice received intraperitoneal injections of 2.0 μl/g.b.w. olive oil twice a week. At each endpoint, serum levels of liver enzymes and liver histology were analyzed.

| The effect of CMC-encapsulated Ad-IL10 on the mouse model of hepatic fibrosis
A total of 40 C57BL/6J mice (male, 6 weeks old) were randomly divided into four groups (10 mice each group). Ten mice were subjected to intraperitoneal injection of olive oil twice a week at 2.0 μl/g.  Mouse cardiac blood collection was carried out as previously described. 37,44 Briefly, mice were first anesthetized by intraperitoneal injection of 3% sodium pentobarbital at 50 mg/kg body weight. An incision was made in upper-middle abdomen across the abdominal and chest cavity to expose the liver and heart, and phlebotomized slowly from the left ventricle until reaching 500 μl for each mouse.
The mice were then euthanized. The collected blood samples were

| Total RNA isolation and touchdownquantitative real-time PCR (TqPCR) analysis
Total RNA from freshly-prepared liver tissues was isolated by using the TRIZOL Reagent (Invitrogen) as described. 45,46 Total RNA was subjected to reverse transcription with hexamer and M-MuLV reverse transcriptase (New England Biolabs). The cDNA products were further diluted and used as PCR templates. Gene-specific qPCR primers were designed by using the Primer3 program (Table S1). TqPCR was carried out by using 2x SYBR Green qPCR Master Mix (Bimake) on a CFX-Connect unit (Bio-Rad Laboratories) as described. 47 All TqPCR reactions were done in triplicate. Gapdh was used as a reference gene.
Quantification of gene expression was carried out by using the 2 ÀΔΔCq method as described. 48

| Hematoxylin and eosin (H&E) analysis and Sirius red staining
The retrieved skin and liver samples were fixed with 4% paraformaldehyde and subjected to paraffin embedding, followed by sectioning.
The slides were deparaffinized and used for H&E staining as previously described. 46,49,50 The sections from the retrieved liver samples were deparaffinized and subjected to Sirius red staining (Picro Sirius Red solution, G1471, Solarbio) as previously reported. 43

| IHC staining
The sections from the above paraffin-embedded skin and liver samples were also deparaffinized and subjected to IHC staining as described. 45,48,51,52 Specifically, the sections were deparaffinized and

| Statistical analysis
Data were analyzed using GraphPad Prism 7 and presented as the mean ± standard deviations (SD). Statistical significance was determined by one-way analysis of variance and the student's t test for the comparisons between groups. A value of p < 0.05 was considered statistically significant. . Interestingly, the Ad-FLuc mixed with 5% CMC did not exhibit higher signal activities than that with 1.25% or 2.5% CMC, indicating that high concentrations of CMC may hamper the release of adenoviral vectors and hence limit transgene expression (Figure 1a, Panel ii), which was further supported by the quantitative analysis of the bioluminescence imaging data ( Figure S1A). Thus, these results suggest that the optimal CMC concentrations for sustained subcutaneous transgene expression may fall between 1.25% and 2.5%. We thus chose to use 1.3% CMC as the optimal concentration for all studies outlined below.
We also tested the duration of in vivo FLuc expression of intrahepatic injection of Ad-FLuc mixed with or without 1.3% CMC.
When the intrahepatic injection was carried out as illustrated in Figure S1B, we found that injections with or without 1.3% CMC exhibited strong FLuc signals at Day 3 ( Figure 1b). However, the Ad-FLuc+1.3% CMC group maintained relatively strong signals at Day 7 (although the signals were not readily detectable at Day 12), while the Ad-FLuc alone group failed to display any FLuc activity under the same imaging condition (Figure 1b, Panel i vs. ii). Collectively, these results demonstrate that optimal concentrations of CMC (e.g., between 1.25% and 2.5%) can effectively prolong adenovirusmediated transgene expression in vivo.  (Figures 3b and S2D). These results strongly suggest that CMC may effectively mitigate adenovirus-induced host acute immune response in vivo.

| CMC alleviates adenovirus-induced chronic inflammatory injury of liver
We next examined the effect of CMC on chronic inflammatory injury after repeated long-term administration of adenovirus to the liver.
Specifically, Ad-GFP mixed with or without 1.3% CMC (wt/vol, in PBS) was intrahepatically injected once every 10 days for 4 and Since chronic inflammatory injury usually activates both cellular immunity and B cell-dependent humoral immunity, 54  Week 4 and IL1β at Week 8, respectively ( Figure S3A). Furthermore, the sirius red staining did not reveal any fibrous hyperplasia in portal area, and the cellular structure of hepatic lobule was relatively normal in both groups at Weeks 4 and 8 ( Figure S3B). Collectively, the above results strongly suggest that CMC may provide a beneficial effect on alleviating chronic inflammatory injury after repeated administration of adenovirus to the liver.

| Intrahepatic administration of CMCencapsulated Ad-IL10 effectively alleviates hepatic fibrosis in a mouse model
While the exact mechanism remains to be fully understood, liver fibrosis is considered as a fibrotic healing response against a chronic injury or insult to the liver. 55,56 Here, we sought to establish an experimental hepatic fibrosis mouse model by intraperitoneal injection of carbon tetrachloride (CCl 4 ). Briefly, C57BL/6J mice were intraperitoneally injected with 2.0 μl/g.b.w. of 20% CCl 4 solution (in olive oil) twice a week for up to 8 weeks as described. 43 The control mice received intraperitoneal injections of 2.0 μl/g.b.w. olive oil twice a week.
Mouse body weight was monitored and exhibited no significant difference between the model group and control group ( Figure S4A). At Week 4 ( Figure S5B). The gross images indicated that while the liver surface in all three groups was rough and granular at both Weeks 4 and 8, the number of white nodules on the liver surface in the Ad-IL10 group and the Ad-IL10 + CMC group was less than that in the fibrosis model group at Week 8 (Figure 5b). H&E staining showed that many inflammatory cells and the balloon-like hepatocytes could be observed in the liver tissues retrieved from the fibrosis model group, to a lesser extent in the Ad-IL10 group, compared with that in the Ad-IL10 + CMC group (Figure 5c). Sirius red staining further confirmed that more collagen deposition and the formation of fibrous septa and pseudolobuli were readily found around the central veins and portal areas of liver tissues in the fibrosis model group, although much less in the Ad-IL10 group at both Weeks 4 and 8 (Figure 5d). Most notably, the number of pseudolobuli and fibrous septa was drastically reduced in the liver tissues retrieved from the Ad-IL10 + CMC group F I G U R E 4 CMC alleviates chronic hepatic inflammation caused by repeated administrations of adenovirus vector in vivo. Ad-GFP with or without 1.3% (wt/vol, in PBS) CMC in 30 μl total volume of PBS were intrahepatically injected once every 10 days; and the mice were sacrificed at Weeks 4 and 8, respectively (a). The retrieved liver tissues were subjected to H&E staining (b). Representative inflammatory cells are indicated with yellow arrows (Â200). The retrieved liver tissues were also subjected to IHC staining with antibodies against CD45 and CD20 (c). Representative positively stained cells are indicated with red arrows (Â400) (c). Representative results are shown at both Weeks 4 and 8 (Figure 5d). These results demonstrate that adenovirus-mediated delivery of IL10, especially when Ad-IL10 was encapsulated with CMC, could effectively alleviate CCl 4 -induced hepatic fibrosis.
It has been well established that hepatic staller cells (HSCs) are the main myofibroblast progenitor cells and key effectors of fibrogenic response. 56 During liver injury, activated HSCs progressively lose their star-shaped morphology and their lipid droplets, and produce abundant extracellular matrix components such as Type I, III, and IV collagens, fibronectin, laminin, and proteoglycans, and proinflammatory mediators, as well as expressing high levels of alpha smooth muscle actin (α-Sma) and tissue inhibitor of metalloproteinase 1 (Timp1) that contribute to the change from adipocytic phenotype to profibrogenic and inflammatory phenotype. The qPCR analysis indicated that while the expression of IL-10 increased, the expression of Type I collagen, α-Sma, and Timp1 significantly decreased both in the Ad-IL10 group and the Ad-IL10 + CMC group, compared with that in the fibrosis model group, although the decrease in the Ad-IL10 + CMC group was more pronounced than that in the Ad-IL10 group ( Figure S5C). Furthermore, the IHC staining analysis revealed the high expression of IL-10 in the Ad-IL10 + CMC group, compared with that in other two groups ( Figure 6a). As expected, the expression of collagen I, α-Sma, and Timp1 was most profoundly down-regulated in the Ad-IL10 + CMC group, compared with that in the fibrosis model group and the Ad-IL10 group (Figures 6b-d and S5D). Collectively, the above results strongly suggest that CMC-encapsulated Ad-IL10 may effectively control the release and production of IL-10 through intrahepatic injection and subsequently alleviate CCl 4 -induced hepatic fibrosis in a mouse model.

| DISCUSSION
Viral vector-mediated gene delivery remains one of the most preferred approaches for liver-directed gene therapy. [1][2][3] Among the viral vectors, although AAV-based gene therapy has recently received a great deal of attention due to AAV's favorable biosafety profile and reduced host immune response, 1,4,5,7,8 Ad vector remains as a vector of choice in many cases including liver-directed gene therapy, because it has at least two advantages over AAV and other viral vectors: it has F I G U R E 5 CMC-encapsulated Ad-IL10 mitigates the development and progression of chronic hepatic fibrosis in a mouse model. A mouse model of chronic hepatic fibrosis was induced by injecting CCl 4 once every 3 days. Ad-IL10 with (IL-10 + CMC group) or without (IL-10 group) 1.3% (wt/vol, in PBS) CMC in 30 μl total volume of PBS were intrahepatically injected once every 10 days into the CCl 4 treated mice (a). The "model only" group received neither CMC and/or Ad-IL10. The mice were sacrificed at Weeks 4 and 8, respectively. Macrophotographs of the gross appearance of the retrieved livers were obtained (b). Representative fibrotic nodules are indicated with white arrows (b). The retrieved liver tissues were subjected to H&E staining (c), and representative inflammatory cells are indicated by yellow arrows while representative hepatic fibrotic damage on hepatocytes and necrosis are indicated with black arrows (Â200) (c). The retrieved liver tissues were further subjected to Sirius red staining (d), and the densely deposited collagens are indicated with blue arrows (Â100) (d). Representative results are shown high gene transfer efficiency, and it is easy to scale up for mass production with high titer. 6,7 However, two major shortcomings for Ad vector as a gene delivery system are the relatively short-term of transgene expression and the solicitation of host immune response. Thus, overcoming these limitations would improve the prospect of using Ad vector as a preferred gene delivery vehicle for gene therapy including liver-directed gene therapy.
In this study, we demonstrated that CMC could effectively prolong Ad-mediated transgene gene expression in vivo, while reducing Ad-induced host immune response, leading to sustained transgene expression. Using whole body optical imaging analysis, we showed that, in the presence of optimal concentrations (e.g., 1.25%-2.5% wt/vol) of CMC, Ad-mediated firefly luciferase (FLuc) expression lasted up to 50 days after a single subcutaneous injection, and at least 7 days after intrahepatic injection. Histologic evaluation and IHC analysis revealed that CMC effectively alleviated Ad-induced host immune response. Interestingly, we found that CMC at higher concentrations (e.g., 5% wt/vol) seemingly decreased FLuc expression, suggesting that high concentrations of CMC may prevent the timely release of Ad vectors and thus limit Ad-mediated transgene delivery efficiency.
While the exact mechanism through which CMC prolongs Ad- In this study, we chose to use CMC as the Ad vector delivery vehicle because CMC is widely available and has been approved for several preclinical and clinical applications such as wound healing dressing, biological imaging, tissue engineering, and controlled drug release. 10,12,13,16 It is conceivable that other chitosan derivatives may also be used as Ad vector delivery carriers. 9,[11][12][13] The chemical versatility of chitosan and its derivatives is reflected by its ability to form a poly-cationic charged polymer at physiological pH, and by its modifiable molecular weight and types of surface modifications, which in turn impact chitosan's chemical and biological properties. 12 In fact, numerous studies have been devoted to modify and optimize CMC and/or other chitosan derivatives. [10][11][12]15 Early studies reported that both linear and branched polyethylenimine (PEI)-graft-chitosan copolymers functioned as efficient DNA/siRNA delivery vehicles both in vitro and in vivo. 63,64 Liang et al. 65 reported that tetradecylquaternized CMC/lipid cation polymeric liposomes had low cytotoxicity and could be used for cancer cell-targeted gene delivery although the in vivo gene delivery efficacy of these copolymers has not been extensively investigated. Lee et al. 66 demonstrated that a novel hybrid polymer, glycol chitosan-methyl acrylate-low molecular weight PEI (GMP), spontaneously assembled with plasmid DNA into nanorods and functioned as a non-viral vector for gene delivery in vitro. It was also shown that the chitosan and poly(ethylene glycol)-grafted (PEGylated) chitosan nanoparticles, encapsulating β-catenin siRNA, effectively decreased β-catenin protein levels in colon cancer cells in vitro. 67 Interestingly, a CMC-modified polyamidoamine dendrimer achieved pH-sensitive drug release in response to tumor microenvironment pH changes. 68 More recently, it has been reported that a reactive oxygen species-sensitive hydrogel with strong free radical scavenging ability was prepared by introducing the thione group into CMC hydrogel, which enhanced CMC's wound healing efficacy. 69 Thus, some of these modified CMC and chitosan derivatives may be further explored as potential Ad delivery carriers.
It is worth noting that many polymers such as PEI, poly(ethylene glycol), poly(l-lysine) (PLL), polyamidoamine dendrimer (PAMAM), and poly (aminoethers) have been exploited to enhance Ad vectormediated gene delivery, mostly by evading host immune response and/or redirecting tropism. [70][71][72] However, our results demonstrate that CMC and potentially other chitosan derivatives may be a superior choice of polymeric helper for Ad gene therapy due to their exceptional properties, including biocompatibility, biodegradability, noncytotoxicity, antimicrobial and anti-inflammation activity, low immunogenicity, inexpensiveness, and accessibility.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials, or available from the corresponding authors upon reasonable request.